Core 3 O-glycan is very short glycan structure which is composed of one N-acetylglucosamine and one N-acetylgalactosamine. The core 3 O-glycan structure is synthesized by core3 synthase (beta 1, 3-N-acetylglucosaminyltransferase 6) using UDP-N-acetylglucosamine as substrate. We revealed that the core 3 O-glycan structure modulates prostate cancer formation and gastrointestinal cell differentiation through regulating the heterodimerization of α2β1 integrin and cell surface expression of differentiation marker proteins respectively. This chapter describes the way to determine the functions of core 3 O-glycan in tumor formation and gastrointestinal cell differentiation.